INTRODUCTION

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Cell cycle progression in eukaryotic cells is orchestrated by cyclin-dependent kinases (Cdks), whose activity is subject to many layers of regulation (for reviews, see references 22, 24, and 26). Cdks (primarily Cdc2 in Schizosaccharomyces pombe and Cdc28p in Saccharomyces cerevisiae) become activated by forming a heterodimeric complex with one of a diverse family of regulatory proteins called cyclins. The activity of cyclin-Cdk complexes can be inhibited by phosphorylation of a tyrosine residue in the Cdk, catalyzed by the Wee1 family of protein kinases. In S. pombe, Cdc2 tyrosine phosphorylation is critical for restraining cyclin-Cdc2 activation until cells reach a threshold size in G₂, at which point dephosphorylation of Cdc2, catalyzed by the phosphatase Cdc25, triggers cyclin-Cdc2 activation and consequent entry into mitosis (reviewed in references 8 and 20).

Cdk tyrosine phosphorylation has also been implicated in the regulation of cell cycle progression by checkpoint controls. Studies in fission yeast have implicated Wee1 and Cdc25 in the checkpoint-mediated G₂ arrest following DNA damage or a block to DNA replication (6, 10, 11, 28). In other species, phosphorylation-site cdc2 mutants have been used to dissect the contribution of inhibitory Cdc2 phosphorylation to these checkpoints. Nonphosphorylatable cdc2 mutants permitted inappropriate cell cycle progression in some cells exposed to DNA damage or DNA replication inhibitors, indicating that Cdc2 phosphorylation was important for the checkpoint response (5, 16, 39). However, severe DNA damage or high doses of DNA replication inhibitors still arrested the cell cycle in most cells containing the cdc2 mutants. This complex result indicated that checkpoint responses can halt the cell cycle through Cdc2 phosphorylation-independent mechanisms as well as through inhibitory Cdc2 phosphorylation (reviewed in reference 20).

In budding yeast, the DNA damage and DNA replication checkpoints do not require tyrosine phosphorylation of Cdc28p (1, 33, 34). It has been suggested that these checkpoints operate at a different stage of the cell cycle in this organism, halting anaphase onset rather than cyclin-Cdc28p activation (38). In contrast, we have described a morphogenesis checkpoint in budding yeast that acts to delay or block cyclin-Cdc28p activation (21, 23, 30, 31). In yeast mutants that fail to polarize the actin cytoskeleton, nuclear division is delayed. This delay helps cells to recover from cytoskeletal insults and to complete bud formation prior to nuclear division, forestalling the generation of binucleate cells. The cell cycle delay was reduced, though not abolished, in cells containing a nonphosphorylatable mutation of CDC28 (21). This finding indicated that much of the checkpoint response was due to Cdc28p tyrosine phosphorylation, but that as in the studies cited above, a Cdk tyrosine phosphorylation-independent mechanism also played some role.

We report here the isolation of a dominant checkpoint-defective mutation of CDC28 in a screen for morphogenesis checkpoint mutants of budding yeast. This allele of CDC28 was more resistant to the action of the morphogenesis checkpoint than was the nonphosphorylatable CDC28 mutation. This result prompted us to reexamine the different components of the checkpoint response, and we found that both the phosphorylation-dependent and the phosphorylation-independent pathways require the Wee1 family kinase, Swe1p. The phosphorylation-site CDC28 mutant was still partly susceptible to inhibition by Swe1p, both in vitro and in vivo. Furthermore, catalytically inactive Swe1p was still able to inhibit Cdc28p in vitro and to sustain a partial checkpoint response. We conclude that the phosphorylation-independent branch of the morphogenesis checkpoint also reflects inhibition of Cdc28p by Swe1p, which can inhibit the nonphosphorylatable CDC28 mutant. Furthermore, our experiments suggest that catalytically inactive Swe1p can respond to a signal from the morphogenesis checkpoint to inhibit Cdc28p.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents. 4',6'-Diamidino-2-phenylindole (DAPI) was purchased from Sigma Chemical Co. (St. Louis, Mo.) and stored as a 1-mg/ml stock solution in H₂O at 20°C. Latrunculin B (Lat-B) was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, Pa.) and stored as a 10 mM stock solution in dimethyl sulfoxide at 20°C. Latrunculin-A (Lat-A), rhodamine-conjugated phalloidin, and Sytox were purchased from Molecular Probes (Eugene, Oreg.). Lat-A was also provided as a generous gift from Phil Crews (University of California, Santa Cruz) and stored as a 20 mM stock solution in dimethyl sulfoxide at 20°C. Rhodamine-conjugated phalloidin was stored as a 200-U/ml stock solution in methanol at 20°C. Mounting medium was made as described elsewhere (27). 5-Fluoroorotic acid (5-FOA) was purchased from Toronto Research Chemicals, Inc. (North York, Ontario, Canada). Anti-Myc antibody 9E10 was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.), and anti-phospho(Tyr15)-Cdc2 antibody was purchased from New England Biolabs (Beverly, Mass.).

Yeast strains and plasmid constructions. The yeast strains used in this study are listed in Table 1. All are in the BF264-15DU background (ade1 his2 leu2-3, 112 trp1-1^a ura3ns). The previously described swe1LEU2 and GAL:SWE1::LEU2 (7), GAP:SWE1::HIS2 (31), GAL:SWE1-myc::URA3 and GAL:MIH1::TRP1 (23), zds1HIS3 (4), GAL:CLB2::LEU2 (34), cdc28-4 (37), zds2URA3, gal:SWE1^K473P-myc::URA3, SWE1^K473P-myc::TRP1, CDC28^Y19F, and CDC28^E12K alleles were introduced into the BF264-15DU background by direct transformation.

zds2URA3

zds2URA3

zds2ura3

zds2URA3

zds1ura3

zds1 zds2

Medium and growth conditions. Strains were grown in rich medium (YEPD [1% yeast extract, 2% Bacto Peptone, 2% dextrose, 0.01% adenine], YEPS [YEPD with 2% sucrose instead of dextrose], or YEPG [YEPD with 2% galactose instead of dextrose]). Cells transformed with plasmids were grown on synthetic dropout medium (0.67% yeast nitrogen base plus 2% dextrose or galactose, supplemented with amino acids). For -factor arrest-release experiments, exponentially growing cells (2 × 10⁶ to 5 × 10⁶ cells/ml) in YEPD were incubated with 20 to 25 ng of -factor per ml for 2 to 3 h, harvested by centrifugation, and resuspended in fresh YEPD to release the -factor-induced cell cycle block. bar1 strains were used in all the -factor arrest experiments. To compare the growth of different strains by spot assay, cells were sonicated and counted with a hemacytometer. For each strain, four 2-µl spots were pipetted onto a YEPD or YEPG plate, each spot containing a total of approximately 1,250, 250, 50, or 10 cells. Strains being compared were pipetted onto the same plate and were incubated at 30°C for the indicated times. For growth rate assays in liquid culture, cells were removed from exponentially growing cultures at five time points over a period of 360 min and sonicated. At least 200 cells from each sample were counted on a hemacytometer from which cell density was calculated. The best-fit line (linear regression) was calculated by using KaleidaGraph (Synergy Software, Reading, Pa.).

Screen to identify suppressors of zds1 zds2. The starting strains, JMY1208 (MATa) and JMY1222 (MAT), were first streaked onto YEPG plates; individual colonies containing 5 × 10⁶ to 1 × 10⁷ cells were resuspended in 100 µl of liquid YEPD and then plated onto separate YEPD plates. A total of approximately 10⁹ cells were plated onto YEPD plates to select for suppressors of the zds1 zds2 mutations. However, this number is conservative because the plated cells divide several times on YEPD before dying. Cell morphology of cells in colonies growing on the YEPD plates was analyzed microscopically, and suppressors with morphologically normal cells were tested for a defect in the morphogenesis checkpoint by using Lat-B as described in Results. Of the eight suppressors isolated that were confirmed to have a morphogenesis checkpoint defect based on the Lat-B assay, four (three from JMY1208 and one from JMY1222) contained the CDC28^E12K allele.

Gap repair of CDC28 alleles. The gap repair plasmid pJM1028 is a pRS315 (32) derivative (LEU2, centromere) containing the CDC28 ORF plus 338 bp upstream and 789 bp downstream. pJM1028 was digested with NdeI (start codon) and HindIII (130 bases upstream of the stop codon), resulting in the deletion of most of the CDC28 ORF. The digested and gel-purified linearized plasmid was transformed into strains isolated from the zds1 zds2 suppressor screen, allowing repair of the gap within the plasmid by homologous recombination with the genomic copy of CDC28. The gap-repaired plasmids were subsequently rescued and the CDC28 ORFs sequenced.

Fluorescence staining, microscopy, and flow cytometry. DNA was stained with DAPI, and F-actin was visualized by staining with rhodamine-conjugated phalloidin as reported previously (23). Cells were viewed on an Axioscope (Carl Zeiss, Inc., Thornwood, N.Y.) equipped with epifluorescence and Nomarski optics. Images were captured with a cooled charge-coupled device camera (Princeton Instruments, Princeton, N.J.). Microscopic images of whole yeast microcolonies were similarly captured. Cells were processed for flow cytometry as described previously (31), using 1 µM Sytox to stain the DNA. The percentages of budded and anaphase cells were quantitated for the same samples as processed for flow cytometry.

p18^CKS1 precipitation and immunoblotting. Strains DLY2626 (CDC28 GAL:SWE1), JMY1390 (CDC28^Y19F GAL:SWE1), and JMY1386 (CDC28^E12K GAL:SWE1) were grown in YEPS and induced to express Swe1p by growth for 4 h after addition of galactose (2%). Cells were then harvested by centrifugation and washed with ice-cold H₂O, and the pellets were frozen at 80°C. Yeast lysates were made as described elsewhere (30). Lysates containing 1 mg of total protein were incubated with 50 µl of p18^CKS1 beads (see below) in 1 ml of binding buffer containing 50 mM sodium phosphate (pH 7.5), 150 mM sodium chloride, 10% glycerol, 10 mM sodium pyrophosphate, 10 mM EDTA, 10 mM EGTA, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 2 µg each of aprotinin, leupeptin, and pepstatin A (Sigma) per ml. Affigel-15 beads (Bio-Rad, Hercules, Calif.) were coupled to p18^CKS1 (1.5 mg of p18/ml of Affigel-15) according to the manufacturer's instructions (p18 was a gift from Mark Watson, The Scripps Research Institute, La Jolla, Calif.). Lysates and beads were mixed by gentle rocking for 4 h at 4°C, and the beads were then washed once in binding buffer and once in binding buffer with 0.3 M potassium chloride, following which the beads were drained and resuspended in 2× sample loading buffer (125 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate [SDS], 50% glycerol, 710 mM -mercaptoethanol, 0.02% bromophenol blue), heated at 95°C for 3 min, diluted twofold with H₂O, and loaded on an SDS-15% polyacrylamide gel. Following electrophoresis, proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, N.H.) and stained with anti-phospho-Cdc2 antibody Tyr 15 (used at 1:1,000 dilution according to the manufacturer's recommendations). The blot was then stripped and reprobed with monoclonal anti-PSTAIRE antibody (used at 1:20,000 dilution of a mouse ascites preparation).

Cdc28p histone H1 kinase assays. (i) Galactose induction. Yeast strains were grown to a density of 0.5 × 10⁷ to 1 × 10⁷ cells/ml in YEPS either at 30°C for RSY16 (GAL:CDC28-GST GAL:CLB2) and RSY17 (GAL:CDC28^Y19F-GST GAL:CLB2) or at 24°C for DLY101 (cdc28-4 control strain), JMY1455 (GAL:SWE1-myc cdc28-4), and JMY1456 (GAL:SWE1^K473P-myc cdc28-4). Cells were induced to express GAL-regulated genes by the addition of galactose to a final concentration of 2% followed by a 3-h incubation of the cultures at their growth temperatures, except for Fig. 6B, in which the JMY1455 and JMY1456 cultures were first shifted to 37°C for 3 h, galactose was added, and the cultures were incubated for an additional 3 h at 37°C. After induction, cells were harvested and washed once with ice-cold H₂O and the cell pellets were stored at 80°C. cdc28-4 strains were used as the source for Swe1p-Myc because in other experiments (data not shown) we found that Swe1p could associate with a Cdc28p-dependent histone H1 kinase activity: Cdc28-4p does not exhibit in vitro kinase activity (37), and so these strains allowed us to circumvent this complicating factor.

(ii) Lysates and immunoprecipitation. Yeast lysates were made as described previously (30). To purify GST-Cdc28p and GST-Cdc28p^Y19F, a 50% glutathione bead slurry (Pharmacia) was added to 500 µg of total protein in 1 ml of Nonidet P-40 (NP-40) lysis buffer (50 mM Tris [pH 7.5], 5 mM EDTA, 1 mM sodium pyrophosphate, 150 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg each of aprotinin, pepstatin, and leupeptin per ml) with 10 mM dithiothreitol (DTT). The bead-lysate mixture was gently mixed for 5 min at 20°C and then given three 1-ml washes in NP-40 lysis buffer with 10 mM DTT. To elute the bound protein, the beads were resuspended in 50 µl of a 25 mM glutathione-50 mM Tris (pH 8)-10 mM DTT solution, gently mixed for 5 min at 20°C, and centrifuged, and the supernatant was removed to a new tube. Immediately before use in the kinase assay, the eluted GST-Cdc28p and GST-Cdc28p^Y19F proteins were diluted 1:1 with reaction buffer (7.5 mM MgCl₂, 20 mM Tris [pH 7.5]). To purify Swe1p-Myc and Swe1p^K473P-Myc, 5 µl of anti-Myc antibody 9E10 was added to 2.5 mg of total protein in 1 ml of NP-40 lysis buffer and mixed for 1 h at 4°C. After addition of 150 µl of a 50% protein A slurry, the tubes were incubated for 1 h at 4°C with mixing. The protein A beads were then washed three times in NP-40 lysis buffer and resuspended in 100 µl of NP-40 lysis buffer.

(iii) Histone H1 kinase assay. Swe1p-Myc-bound beads (10 µl) and eluted GST-Cdc28p 1 µl were added to 19 µl of reaction buffer and incubated for 20 min at 20°C (GST-Cdc28p was not added to the kinase assay in Fig. 7A). After addition of 15 µl of a kinase reaction cocktail, the mixture was incubated at 30°C for 30 min. The final kinase reaction mixture contained 7.5 mM MgCl₂, 20 mM Tris (pH 7.5), 2 µg of histone H1, 0.1 nM ATP, 10 µCi of [-³²P]ATP (3,000 Ci/mmol), 10 µl of Swe1p-Myc (or Swe1p^K473P-Myc) beads, and 1 µl of GST-Cdc28p (or GST-Cdc28p^Y19F) in a total volume of 45 µl. The reaction was stopped with the addition of 15 µl of sample loading buffer, and the mixture was heated at 100°C for 5 min. Then 20 µl of each reaction mixture was loaded onto an SDS-11% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie blue to visualize the histone H1, boiled for 10 min in 5% trichloroacetic acid to remove remaining unincorporated nucleotide, and dried on Whatman 3MM paper. After exposure to film, the stained bands corresponding to histone H1 were cut out of the gel and counted on a scintillation counter. Cdc28p activity in the presence of Swe1p was normalized to the activity in the absence of Swe1p (following subtraction of background counts).

	RESULTS

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Isolation of morphogenesis checkpoint-defective mutants. We devised a two-step genetic screen for mutants with a defective morphogenesis checkpoint. The first step depends on the phenotype of zds1 zds2 mutant strains. Bi and Pringle (4) identified ZDS1 in a screen for regulators of Cdc42p and demonstrated that a GST-Zds1p fusion protein was concentrated at presumptive bud sites and at the tip of small buds, suggestive of a role in cell morphogenesis. A homolog of ZDS1, called ZDS2, was identified; the zds1 zds2 mutant strain grows very slowly, is delayed in G₂, and displays morphological abnormalities that include elongated buds. The exact role of the ZDS genes remains obscure, particularly since they have been identified independently by multiple investigators in diverse screens (zds stands for "zillion different screens" [cited in reference 4]). However, Wang and Burke (36) reported that the poor growth and morphologic abnormalities of zds1 zds2 mutants could be rescued by expression of the CDC28^Y19F allele that cannot be phosphorylated at Y19. They suggested that one primary defect in zds1 zds2 mutants was a mild morphogenesis defect and that this defect triggered the morphogenesis checkpoint to cause a prolonged G₂ delay through Cdc28p Y19 phosphorylation (36). In this scenario, the morphogenesis checkpoint was in fact compromising the growth of an otherwise only mildly defective strain.

View larger version (70K): [in this window] [in a new window]   FIG. 1.   Rescue of zds1 zds2 strains by overexpression of MIH1 or deletion of SWE1. (A) Spot assays of wild-type (DLY1), zds1 zds2 GAL:MIH1 (JMY1208), and zds1 zds2 swe1 (JMY1207) on YEPD (dextrose) and YEPG (galactose) after 2 days of growth at 30°C. Both the overexpression of MIH1 and deletion of SWE1 rescue the growth defect of a zds1 zds2 mutant. (B) Colony morphology of the above strains after 1 day of growth on dextrose or galactose-containing media at 30°C. Bar = 20 µm.

swe1

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Cell cycle arrest in Lat-B-treated cells. (A) F-actin organization in wild-type (DLY1) cells exposed to 0 and 100 µM Lat-B. Actin was visualized by staining cells with rhodamine-conjugated phalloidin. In the cells treated with 100 µM Lat-B, cables are absent and the actin patches are depolarized. Bar = 5 µm. (B) Kinetics of nuclear division for wild-type (WT; DLY1) and swe1 (DLY1028) cells after release from -factor-induced G1 arrest into fresh medium containing 100 µM Lat-B. All cultures were grown in YEPD at 30°C.

The dominant checkpoint-defective mutants all contain a single mutation in CDC28. Since Swe1p is known to inhibit cyclin-Cdc28p complexes through phosphorylation of Cdc28p Y19, it seemed possible that the dominant mutants we obtained contained alterations (e.g., at the Y19 position) in the CDC28 gene. One of the four independent dominant mutants was crossed to each of the others and to a strain containing a cdc28-4 temperature-sensitive allele. The resulting diploids were sporulated, and tetrads were dissected to determine whether the mutants were in linked genes. No wild-type recombinants were obtained in 22 tetrads from these crosses, suggesting that all four strains contained mutations in linked genes, possibly all in the CDC28 gene. To test this, we rescued the CDC28 gene from the genome of each mutant strain (as well as a wild-type parent strain) by using gap repair (see Materials and Methods). The plasmid-borne CDC28 alleles from the mutant strains were all able to suppress the poor growth and morphologic abnormalities of the parent zds1 zds2 strain and conferred a morphogenesis checkpoint defect (see Fig. 4 and 5), confirming that CDC28 had been mutated in each of the strains.

View larger version (56K): [in this window] [in a new window]   FIG. 3.   A CDC28E12K mutant has a normal growth profile but is resistant to SWE1 overexpression. (A) Sequence homology of the amino terminus of S. cerevisiae (Sc) Cdc28p with the other Cdks in S. cerevisiae and with S. pombe and human (Hs) Cdks. The conserved tyrosine (Y) residue phosphorylated by Wee1 family kinases and the glutamic acid (E) residue mutated to lysine (K) in the CDC28 mutants isolated from the zds1 zds2 suppression screen are shaded. S. cerevisiae Kin28p, Ctk1p, and Ssn3p and human Cdk4 do not have a glutamic acid at this position; however, the human Cdk4 does have an adjacent glutamic acid residue that is boxed but not shaded. Ctk1p and Ssn3p have large amino-terminal regions, and the sequence shown begins at residue 179 for Ctk1p and 71 for Ssn3p. (B) Growth rates of exponentially proliferating wild-type and CDC28E12K strains. (C) Flow cytometric analysis of the DNA content of exponentially proliferating wild-type (WT) and CDC28E12K cells. (D) Percentage of budded and anaphase cells, calculated from microscopic examination of the same cells stained for flow cytometric analysis in panel C. (E) Cell morphology of exponentially growing wild-type and CDC28E12K cells. Bar = 5 µm. (F) Cell morphology of wild-type, GAL:SWE1, and GAL:SWE1 CDC28E12K strains 4 h after addition of galactose to cells growing in YEPS medium. Bar = 5 µm. The strains used were DLY1 (wild type), JMY1380 (CDC28E12K), DLY2626 (GAL:SWE1), and JMY1386 (CDC28E12K GAL:SWE1). (G) Tyrosine phosphorylation of Cdc28p alleles upon overexpression of Swe1p. GAL:SWE1 strains containing wild-type (DLY2626), Y19F (JMY1390), and E12K (JMY1386) alleles of Cdc28p were induced to overexpress Swe1p by growth for 4 h after addition of galactose to cells growing in YEPS medium. p18Cks1 beads were then used to precipitate the different Cdc28p proteins, which were immunoblotted with anti-phospho-Cdc2 antibody to detect tyrosine-phosphorylated Cdc28p and anti-PSATIRE antibodies to detect total Cdc28p. All cultures were grown at 30°C and, unless otherwise indicated, were grown in YEPD.

CDC28^E12K does not affect cell cycle progression during the unperturbed cell cycle. To analyze the phenotype of the CDC28^E12K mutant in more detail, we constructed a strain in which the wild-type CDC28 gene, at its normal locus, was replaced with the E12K allele (see Materials and Methods). This strain allowed us to determine whether the allele conferred any recessive cell cycle phenotype. Cells containing CDC28^E12K grew at the same rate as wild-type cells (Fig. 3B), displayed similar cell cycle profiles by flow cytometry (Fig. 3C), and exhibited a normal cell morphology (Fig. 3E). In addition, asynchronous populations from these strains displayed similar percentages of budded and anaphase cells (Fig. 3D). Thus, the E12K allele has no detectable effect on the unperturbed cell cycle of budding yeast.

CDC28^E12K renders cells resistant to Swe1p overexpression. Both the morphogenesis checkpoint defect exhibited by CDC28^E12K and its lack of a cell cycle defect in the unperturbed cycle are reminiscent of the CDC28^Y19F mutant that cannot be phosphorylated by Swe1p. If the E12K allele is simply insensitive to inhibition by Swe1p, then strains containing this allele should be resistant to the G₂ arrest caused by Swe1p overexpression. To test this, strains containing GAL-SWE1 and either wild-type or E12K CDC28 were induced to express Swe1p by addition of galactose to the medium. While the cells containing wild-type CDC28 formed elongated buds characteristic of G₂ arrest on galactose, the cells containing the E12K allele were resistant to the GAL-SWE1 and proliferated with normal cell morphology (Fig. 3F). In principle, this resistance might arise in two ways: Swe1p may be unable to phosphorylate Cdc28p^E12K, or Cdc28p^E12K might retain activity even following phosphorylation on Y19. To distinguish between these possibilities, we examined whether Cdc28p^E12K became phosphorylated at Y19 following overexpression of Swe1p (Fig. 3G). We used an anti-phospho-Cdc2/Cdc28p antibody to immunoblot Cdc28p proteins isolated from Swe1p-overexpressing strains by using p18^CKS1 beads (see Materials and Methods). This reagent detected wild-type Cdc28p but not Cdc28p^Y19F, confirming its specificity for Y19-phosphorylated Cdc28p (Fig. 3G). Cdc28p^E12K was not detected by the anti-phospho-Cdc2/Cdc28p antibody, although the protein was present, as evidenced by immunoblotting the same membrane with anti-PSTAIRE antibody (Fig. 3G). Thus, the E12K substitution confers resistance to Swe1p by reducing or eliminating Y19 phosphorylation.

Comparison of the CDC28^Y19F and CDC28^E12K mutants. The repeated independent isolation of the CDC28^E12K mutation and the failure to isolate a CDC28^Y19F mutation (which would require a single A-to-T change) seemed surprising. To determine whether this was a chance result or whether there might be phenotypic differences between the E12K and Y19F alleles, we transformed these alleles on a low-copy-number plasmid into the strains used for the genetic screen. In the zds1 zds2 strain (JMY1222), both alleles were able to suppress the slow-growth defect, but only the CDC28^E12K allele effectively suppressed the morphologic abnormalities (Fig. 4). zds1 zds2 mutants containing the CDC28^Y19F allele continued to display abnormal morphologies including elongated buds, although to a lesser degree than the parent strain (Fig. 4). This result explains our failure to isolate such alleles by using our stringent primary screen criteria.

View larger version (75K): [in this window] [in a new window]   FIG. 4.   The CDC28E12K allele fully rescues the zds1 zds2 mutant, while the CDC28Y19F allele only partially rescues cell morphology. (A) Spot assays of a zds1 zds2 GAL:MIH1 strain (JMY1222) transformed with a centromere plasmid (pRS316) carrying CDC28 or the CDC28E12K or CDC28Y19F alleles, grown on YEPG and YEPD at 30°C for 3 days and 2 days, respectively. Both the CDC28E12K and CDC28Y19F alleles rescue the growth defect in a zds1 zds2 strain. (B) Colony morphology of the above strains after 1 day of growth on dextrose-containing medium at 30°C. Bar = 20 µm.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   Morphogenesis checkpoint-induced delay of nuclear division in CDC28Y19F and CDC28E12K mutants. Strains were released from -factor-induced cell cycle arrest into fresh medium containing 50 µM Lat-A. At the indicated times, aliquots were fixed and subsequently stained with DAPI to visualize DNA; 200 cells were scored in each sample. Strains used were DLY1 (SWE1 CDC28), JMY1362 (SWE1 CDC28E12K), JMY1364 (SWE1 CDC28Y19F), DLY1028 (swe1 CDC28), JMY1365 (swe1 CDC28E12K), and JMY1367 (swe1 CDC28Y19F).

Cells lacking Swe1p have a more severe checkpoint defect than cells containing the checkpoint-defective CDC28 alleles. In previous studies, we had interpreted the residual checkpoint-induced G₂ delay in cells containing the CDC28^Y19F allele to indicate the existence of a second pathway capable of inhibiting Cdc28p in response to morphogenesis insults (21). The reduced G₂ delay in cells containing the CDC28^E12K allele might therefore indicate that this mutant was resistant both to the Swe1p-mediated phosphorylation pathway and the putative second pathway. To address this issue further, we compared the kinetics of nuclear division in cells lacking Swe1p versus cells containing nonphosphorylatable Cdc28p (Fig. 5). Surprisingly, swe1 cells underwent nuclear division even earlier than the cells containing the CDC28^E12K allele (Fig. 5). In addition, the swe1 cells underwent nuclear division at the same time regardless of which CDC28 allele was present (Fig. 5). This finding suggests that both the E12K and Y19F forms of Cdc28p are still sensitive (to a reduced extent) to inhibition by Swe1p: Cdc28p^E12K is more resistant to inhibition by Swe1p than Cdc28p^Y19F.

Phosphorylation-independent inhibition of Cdc28p by Swe1p. Booher and colleagues have reported that Swe1p can inhibit Cdc28p^Y19F in vitro, in a manner that is sensitive to dilution (7). We confirmed the observation that Swe1p could inhibit Cdc28p^Y19F as well as wild-type Cdc28p (Fig. 6A). In this experiment, GST-Cdc28p/Clb2p complexes were isolated from yeast cells as described in Materials and Methods and mixed with Swe1p immunoprecipitates in the absence of ATP to allow binding. Histone H1 and [-³²P]ATP were then added, and histone H1 kinase activity was monitored. Under these conditions, Swe1p inhibited wild-type and Y19F Cdc28p to similar extents (Fig. 6A).

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Phosphorylation-independent inhibition of Cdc28p by Swe1p in vitro. (A) Immunoprecipitates containing Swe1p from strains expressing (WT [wild type]) or not expressing () GAL:SWE1-myc were mixed as indicated with GST-Cdc28p/Clb2p (WT) or GST-Cdc28pY19F/Clb2p (Y19F) complexes isolated from yeast and subsequently assayed for Cdc28p-dependent histone H1 kinase activity as described in Materials and Methods. In the autoradiograph of radioactive histone H1, histone bands were excised from the gel and the incorporated radioactivity was quantitated and plotted above the lanes. Activity was normalized so that the activity of wild-type Cdc28p in the absence of Swe1p was made equal to 1. Swe1p inhibited wild-type and Y19F Cdc28p to similar extents. (B) Assays were performed as for panel A but with immunoprecipitated Swe1p (WT) or Swe1pK473P (K473P). The inset shows a Western blot of the Myc-tagged Swe1p proteins from the same immunoprecipitates used for the kinase assay. Wild-type and kinase-dead forms of Swe1p inhibited CDC28p to similar extents.

Endogenous levels of Swe1p^K473P provide a partial checkpoint response. The inhibition of Cdc28p by catalytically inactive Swe1p in vitro suggested that catalytically inert Swe1p might be able to function similarly in vivo and provide a partial checkpoint response. To test this, we generated a strain in which Myc-tagged Swe1p^K473P was expressed at the SWE1 genomic locus from its own promoter (see Materials and Methods). Cells containing wild-type Swe1p, catalytically inactive Swe1p^K473P, or no Swe1p were then synchronized, and their response to actin perturbation by Lat-A was monitored (Fig. 7). As expected, cells containing wild-type Swe1p underwent a G₂ arrest, while cells lacking Swe1p did not. Cells containing Swe1p^K473P displayed an intermediate response, delaying but not blocking nuclear division (Fig. 7). This demonstrates that noncatalytic inhibition of Cdc28p by Swe1p can play a role during a physiological checkpoint response.

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Morphogenesis checkpoint-induced delay of nuclear division in a swe1K473P mutant. Strains were released from -factor-induced cell cycle arrest into fresh medium containing 50 µM Lat-A. At the indicated times, aliquots were fixed and subsequently stained with DAPI to visualize DNA; 200 cells were scored in each sample. Strains were DLY1 (WT [wild type]), DLY1028 (swe1), and JMY1428 (swe1K473P-myc).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Genetic screen for morphogenesis checkpoint mutants. We report the results of a two-part screen for spontaneous mutants that abrogate the morphogenesis checkpoint in budding yeast. The first step was a selection for suppressors of the zds1 zds2 slow-growth phenotype, predicated on the assumption that the cause of slow growth in these mutants was an inappropriately strong checkpoint arrest. Consistent with this assumption (first suggested by Wang and Burke [36]), we showed that the slow growth and abnormal morphology of these mutants were suppressed by deletion of SWE1 or overexpression of MIH1. The second step was a direct screen for mutants that failed to arrest the cell cycle following perturbation of the actin cytoskeleton. This screen gave rise to two classes of mutants that are expected to arise at very low frequency; the first class inactivated both of the copies of SWE1 present in the starting strain, while the second class consisted of a single dominant checkpoint-defective allele of CDC28.

CDC28^E12K is resistant to inhibition by Swe1p. A further surprise stemming from our screen was that all four independent dominant mutations in CDC28 caused the same amino acid change, E12 to K. Phenotypic analysis demonstrated that a strain containing this mutant grew normally under nonperturbing conditions but was largely unable to delay the cell cycle in response to the morphogenesis checkpoint. In addition, the E12K mutant rendered cells resistant to overexpression of Swe1p, which arrests cells containing the wild-type Cdc28p in G₂. Finally, we found that Cdc28p^E12K was not phosphorylated at Y19 upon overexpression of Swe1p.

Conservation of the E12 residue among Cdks. A comparison of the amino-terminal sequences in different Cdks revealed that the E12 residue was not highly conserved in general but was present in all Cdks known to be subject to inhibition by Wee1 family kinases (Cdc28p, Cdc2, and Cdk2 [Fig. 3A]). Given our data, we suggest that this is a key residue for recognition by Wee1 family enzymes. Interestingly, among the five budding yeast Cdks only Pho85p shares this residue with Cdc28p (Fig. 3A). This raises the possibility that Pho85p might also be a Swe1p target and suggests that the other yeast Cdks (Kin28p, Ctk1p, and Ssn3p/Ume5p/Srb10p) are not Swe1p targets.

Swe1p inhibits the nonphosphorylatable Cdc28p^Y19F. Based on previous findings with strains containing the CDC28^Y19F allele, we concluded that in addition to cell cycle arrest mediated by phosphorylation of Y19 in Cdc28p, the morphogenesis checkpoint induced a second phosphorylation-independent arrest pathway (21). The data presented in this report suggest that this second pathway is not distinct, but rather is also mediated by Swe1p, which can still inhibit the Y19F form of Cdc28p. Further, we found that a mutant Swe1p predicted to lack kinase activity was still able to inhibit Cdc28p. Therefore, a single pathway involving Swe1p-mediated Cdc28p inhibition fully accounts for the cell cycle delay (or arrest) in response to the morphogenesis checkpoint.

Catalytically inactive Swe1p responds to the morphogenesis checkpoint. Remarkably, we found that Swe1p lacking catalytic activity, when expressed at single copy, could significantly delay the cell cycle in vivo during a physiological checkpoint response. In contrast, even GAL-mediated overexpression of the catalytically inactive Swe1p did not significantly delay the cell cycle in cells not exposed to an actin perturbation (data not shown). This suggests that the checkpoint must enhance Swe1p effectiveness (even for the catalytically inert mutant), and not simply Swe1p abundance. Thus, the recently described stabilization of Swe1p in response to checkpoint insults (30) cannot fully account for increased Swe1p activity during a checkpoint response.

Implications for other systems. Our findings have far-reaching implications for many studies in other systems that have utilized nonphosphorylatable Cdc2 mutants on the assumption that they completely bypass the action of Wee1-related kinases. In Aspergillus, cells expressing a nonphosphorylatable Cdc2 mutant failed to delay mitosis when incubated in low doses of hydroxyurea but were still able to arrest in response to high doses of hydroxyurea, suggesting that the checkpoint induced a separate pathway (possibly involving BimE [39]). In Xenopus, addition of cyclin B complexed with a nonphosphorylatable Cdc2 mutant to egg extracts promoted entry into mitosis of interphase extracts, but not checkpoint-arrested extracts (18), suggesting that the DNA replication checkpoint induced a separate pathway (possibly involving a Cdc2 inhibitor [18, 19]). In human cells, inhibitory Cdc2 phosphorylation was important for the DNA damage and DNA replication checkpoints (5, 16), but cells containing a nonphosphorylatable Cdc2 mutant could still delay mitosis when these checkpoints were maximally induced (16), suggesting that these checkpoints induced a separate pathway (possibly preventing the nuclear localization of cyclin-Cdc2 complexes [16, 17, 35]). In all of these cases, our findings suggest an alternative possible interpretation in which a single pathway involving Wee1 like kinases may have inhibited the nonphosphorylatable Cdc2 mutant.

Conclusion. We have isolated a novel morphogenesis checkpoint-defective allele of CDC28 in budding yeast. Analyses using this mutant and a nonphosphorylatable CDC28 mutant show that Swe1p can inhibit Cdc28p without phosphorylating Cdc28p, under physiological conditions. We conclude that a single Swe1p-dependent pathway mediates arrest by this checkpoint, contrary to our previous conclusion (21) indicating a bifurcated pathway. Similar bifurcated pathways (reviewed in reference 20), proposed to account for the action of DNA replication and DNA damage checkpoints in other systems, must now be reevaluated.